The present invention relates to a test medium and method for the detection, quantification, identification and/or differentiation of biological materials in a sample which may contain a plurality of different biological materials.
Bacteria are the causative factor in many diseases of humans, higher animals and plants, and are commonly transmitted by carriers such as water, beverages, food and other organisms. The testing of these potential carriers of bacteria is of critical importance and generally relies on xe2x80x9cindicator organisms.xe2x80x9d Borrego et al., Microbiol. Sem. 13:413-426, (1998). For example, Escherichia coli (E. coli) is a gram negative member of the family Enterobacteriaceae which is part of the normal intestinal flora of warm blooded animals, and its presence indicates fecal contamination (e.g., raw sewage). Even though most strains of E. coli are not the actual cause of disease, their presence is a strong indication of the possible presence of pathogens associated with intestinal disease, such as cholera, dysentery, and hepatitis, among others. Consequently, E. coli has become a prime indicator organism for fecal contamination, and as a result, any method which differentiates and identifies E. coli from other bacteria is very useful.
Others members of the family Enterobacteriaceae, commonly referred to as xe2x80x9cgeneral coliforms,xe2x80x9d especially the genera Citrobacter, Enterobacter and Klebsiella, are also considered to be significant indicator organisms for the quality of water, beverages and foods. Therefore, tests to identify and differentiate general coliforms from E. coli are also very useful. Also, various species of the genus Aeromonas have been shown to not only be potential pathogens, but to have a correlation to other indicator organisms (Pettibone et al., J. App. Microbiol. 85:723-730 (1998)). Current test methods to identify, separate and enumerate Aeromonas spp. from the very similar Enterobacteriaceae have been lacking and most of the current methods utilizing enzyme substrates do not separate Aeromonas spp. from Enterobacteriaceae due to their almost identical biochemical profiles. Any method that depends upon the identification of general coliforms by means of a xcex2-galactosidase substrate either does not differentiate Aeromonas spp. from general coliforms or eliminates Aeromonas from the sample by the use of specific inhibitors (antibiotic such as cefsulodin). Brenner et al., Appl. Envir. Microbio. 59:3534-44 (1993). They do not differentiate, identify and enumerate Aeromonas along with E. coli and general coliforms. Landre et al., Letters Appl. Microbiol. 26:352-354(1998). Improved test methods to effectively identify, separate and enumerate such bacterial types are needed, and there is a continuing search for faster, more accurate, easier to use and more versatile test methods and apparatus in this area.
Numerous test methods have been utilized to determine, identify and enumerate one or more indicator organisms. Some of these test methods only indicate the presence or absence of the microorganism, while others also attempt to quantify one or more of the particular organisms in the test sample. For example, a qualitative test referred to as the Presence/Absence (or P/A) test, may be utilized to determine the presence or absence of coliforms and E. coli in a test sample. A test medium including the xcex2-galactosidase substrate O-nitrophenyl-xcex2-D-galactopyranoside (ONPG), and the xcex2-glucuronidase substrate 4-methyl-umbrelliferyl-xcex2-D-glucuronide (MUG), is inoculated with the test sample. To differentiate the general coliforms from E. coli, this test relies on the fact that generally all coliforms produce xcex2-galactosidase, whereas only E. coli also produces xcex2-glucuronidase in addition to xcex2-galactosidase. If any coliforms are present (including E. coli), the broth medium turns a yellow color due to the activity of the galactosidase enzyme on the ONPG material, causing the release of a diffusible yellow pigment. If E. coli is present, the broth medium will demonstrate a blue fluorescence when irradiated with ultraviolet rays, due to the breakdown of the MUG reagent with the release of the fluorogenic dye caused by the production of the glucuronidase enzyme. These reactions are very specific, and allow the presence of both coliforms in general, as well as E. coli to be identified in a single sample. A disadvantage of this test is that it is not directly quantitative for either bacterial type, since both reagents produce diffusible pigments. A second disadvantage is that there may a false positive coliform reaction if Aeromonas spp. are present in the test sample. This has been shown to be possible even when there are inhibitors present to supposedly prevent this from occurring (Landre et al., Letters Appl. Microbiol. 26:352-354 (1998)). The test also requires specific equipment for producing the ultraviolet rays. Further, this test may only be used to detect coliforms and E. coli. Other important microorganisms, such as the strain E. coli 0157 which is glucuronidase negative, are not detected, nor are other non-galactosidase-glucuronidase producing microorganisms.
The Violet Red Bile Agar (VRBA) method has been used to determine the quantity of both coliform and E. coli in a test sample. The test medium used in this method includes bile salts (to inhibit non-coliforms), lactose and the pH indicator neutral red. As coliforms (including E. coli) grow in the medium, the lactose is fermented with acid production, and the neutral red in the area of the bacterial colony becomes a brick red color. The results of this test are not always easy to interpret, and in order to determine the presence of E coli, confirming follow-up tests, such as brilliant green lactose broth fermentation, growth in EC broth at 44.5xc2x0 C. and streaking on Eosin Methylene Blue Agar (EMBA), must be performed.
The Membrane Filter (MF) method utilizes micropore filters through which samples are passed so that the bacteria are retained on the surface of the filter. This method is used most often when bacterial populations are very small, and a large sample is needed to get adequate numbers. The filter is then placed on the surface of a chosen medium, incubated, and the bacterial colonies growing on the membrane filter surface are counted and evaluated. This method is widely used and provides good results when combined with proper reagents and media A disadvantage of this method is that it is expensive and time-consuming. It also does not work well with solid samples, or samples with high particulate counts. The MF method can be used in conjunction with the inventive method described in this application.
The m-Endo method is also used to determine the quantity of E. coli and general coliforms and is an official USEPA approved method for testing water quality. The medium is commonly used with a membrane filter and E. coli and general coliform colony forming units (CFU) grow as dark colonies with a golden green metallic sheen. Due to a proven high rate of false positive error, typical colonies must be confirmed by additional testing. Standard Methods for the Examination of water and Wastewater, 20th Edition, 9-10 and9-60 (1998).
Other tests, such as the Most Probable Number (MPN), utilize lactose containing broths (LST, BGLB, EC) to estimate numbers of general coliforms and E. coli, but have also been shown to have high rates or error as well as being cumbersome and slow to produce results. Evans et al., Appl. Envir. Microbiol 41:130-138 (1981).
The reagent 5-bromo-4-chloro-3-indolyl-xcex2-D-galactopyranoside (X-gal) is a known test compound for identifying coliforms. When acted on by the xcex2-galactosidase enzyme produced by coliforms, X-gal forms an insoluble indigo blue precipitate. X-gal can be incorporated into a nutrient medium such as an agar plate, and if a sample containing coliforms is present, the coliforms will grow as indigo blue colonies. X-gal has the advantage over the compound ONPG, described above, in that it forms a water insoluble precipitate rather than a diffusible compound, thereby enabling a quantitative determination of coliforms to be made when the test sample is incorporated into or onto a solidified medium, or when coliform colonies grow on the surface of a membrane filter resting on a pad saturated with a liquid medium or on a membrane filter resting on a solid medium. Further, it does not require the use of ultraviolet light.
A similar compound, 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide (X-gluc) is a known test compound for identifying E. coli. When acted on by the xcex2-glucuronidase enzyme produced by most E. coli, X-gluc forms an insoluble indigo blue precipitate. X-gluc has the advantage over the compound MUG, described above, in that it forms a water insoluble precipitate, rather than a diffusible compound, thereby enabling a quantitative determination of E. coli to be made when the test sample is incorporated into or onto a solidified medium. X-gluc and its ability to identify E. coli are described in Watkins, et al., Appl. Envir. Microbiol. 54:1874-1875 (1988). A similar compound, indoxyl-xcex2-D-glucuronide, which also produces sharp blue colonies of E. coli, was described in Ley, et al., Can. J. Microbiol. 34:690-693 (1987).
Although X-gal and X-gluc are each separately useful in the quantitative determination of either coliforms (X-gal) or E. coli (X-gluc), these indicator compounds have the disadvantage that they each contain the same chromogenic component. Therefore, they cannot be used together to identify and distinguish both E. coli and general coliforms in a single test with a single sample, since they both generate identically hued indigo blue colonies. A person using both reagents together would be able to quantitatively identify the total number of coliforms, the same as if X-gal were used alone, but would not be able to indicate which of the colonies were E. coli and which were other coliforms besides E. coli. 
A recently developed and highly commercially successful test method and test medium for quantitatively identifying and differentiating general coliforms and E. coli in a test sample is described in U.S. Pat. Nos. 5,210,022, and 5,393,662, both of which share common inventorship with the present application and which are hereby incorporated by reference. This method and test medium improves upon prior art methods by allowing the quantitative identification of general coliforms and E. coli in a single sample. Additional confirmatory tests are not necessary. The test sample is added to a medium containing a galactosidase substrate, such as 6-chloroindolyl-xcex2-D-galactoside, and a xcex2-glucuronidase substrate, such as 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide (X-gluc). The xcex2-galactosidase substrate is capable of forming a water insoluble precipitate of a first color upon reacting with xcex2-galactosidase, and the xcex2-glucuronidase substrate is capable of forming a water insoluble precipitate of a second color, contrasting with the first color, upon reacting with xcex2-glucuronidase. As a result, general coliforms may be quantified by enumerating the colonies of the first color (having xcex2-galactosidase activity), and E. coli may be quantified by enumerating the colonies of the second color (having both xcex2-galactosidase and xcex2-glucuronidase activity). This technology has been widely copied.
Another recently developed test method and apparatus provides excellent results for the differentiation and identification of general coliforms, E. coli and E. coli 0157 strains and non-coliform Enterobacteriaceae. The method and test medium are described in U.S. Pat. No. 5,726,031, which shares common inventorship with the present application, and which is hereby incorporated by reference.
A certain class of substrates, referred to herein as xe2x80x9cnonchromogenic,xe2x80x9d have been used to detect various microorganisms. A dipslide for detecting E. coli using hydroxy-quinoline-xcex2-D-glucuronide is disclosed by Dalet et al., J. Clin. Microbiol, 33(5):1395-8 (1995). Similarly, a technique for detection of E. coli in an agar-based medium using 8-hydroxyquinoline-xcex2-D-glucuronide is disclosed by James et al., Zentralbl Bakteriol Mikrobiol Hyg [A], 267(3):316-21 (1988).
It is desirable to further improve the distinguishing colors generated in the test media. That is to say, in prior art test media which detect and distinguishing two biological entities, confusion may arise between the two colors which show in the media.
Further, it is desirable to be able to identify and differentiate other closely related organisms, such as members of the families Aeromonaceae, Vibrionaceae, and Salmonella and Shigella spp. For example, the genus Aeromonas is closely related to coliforms and gives an almost identical biochemical test pattern. Further, the genus Vibrio is also an important type of bacteria that grows under the same general conditions as coliforms. It is known to distinguish Aeromonas colonies from general coliforms by testing all colonies in a given sample for the presence of cytochrome oxidase. Undesirably, however, this requires another set of tests. Further, Aeromonas is common in water and food, and it is commonly indicated in test samples as general coliforms, which results in high a false positive error for general coliforms by most current test methods. The Aeromonas can be prevented from interfering with the coliform results by adding certain antibiotics to the medium. However, the antibiotic amounts added must be carefully controlled. Further, the antibiotics which have been conventionally used have short life spans in the media so that they lose their potency quickly in other than a frozen condition. It may often be desirable to be able to culture, identify and enumerate Aeromonas spp. which cannot be done if they are inhibited.
Further, in those cases where it is desirable to inhibit Aeromonas, it is desirable for a better method of so doing, one in which the shelf life of the medium is not appreciably reduced by the inclusion of an inhibitor.
Additionally, it is also desirable to distinguish strains of Salmonella and Shigella from E. coli, general coliforms and Aeromonas.
The present invention overcomes the disadvantages of prior art methods by providing a test method and medium for quantitatively or qualitatively identifying and differentiating biological entities in a test sample that may include a plurality of different biological entities.
The present invention introduces the use of xe2x80x9cnonchromogenicxe2x80x9d substrates to enhance the distinction among multiple colors produced by distinct biological entities present in the inventive test medium. Unexpectedly, it has been discovered that other xe2x80x9cchromogenicxe2x80x9d substrates present in the inventive test medium do not interfere with the substantially black color achieved with the nonchromogenic substrate. That is to say, so long as a given biological entity is responsive to the nonchromogenic substrate, aggregations thereof present in the test medium will show as a substantially black color-independent of whether such biological entity is responsive to one, two or more chromogenic substrates which are also present in the medium. The present invention exploits this hitherto unexplored property of nonchromogenic substrates.
In one form thereof, the present invention provides a test medium for detecting, identifying and qualifying or quantifying first and second biological entities. The test medium includes a nutrient base medium including ions of a salt, a chromogenic substrate and a nonchromogenic substrate. The first biological entity is responsive to the nonchromogenic substrate whereas the second biological entity is responsive to the chromogenic substrate. In this test medium, aggregations of the first biological entity present in the test medium are substantially black and aggregations of the second biological entity present in the test medium are a second color, the second color being distinguishable from the substantially black.
In a preferred form, the inventive test medium accounts for the first biological entity being responsive to the chromogenic substrate in addition to the nonchromogenic substrate. In such event, aggregations of the first biological entity present in the test medium will nonetheless show as substantially black.
Significantly, even though the aggregations of the first biological entity are responsive to both the first and second substrates in the preferred form, these aggregations still show as substantially black in the test medium. That is, the chromogenic substrate does not interfere with the substantially black color. Advantageously, this property of nonchromogenic substrates allows several different biological entities to be identified and differentiated in a single medium, aggregations of each biological entity having a visually distinguishable color.
In another preferred form of the above-described inventive medium, the medium further includes the antibiotic nalidixic acid to inhibit the growth of Aeromonas, spp. Advantageously, it has been found that nalidixic acid, as compared with cefsulodin, does not significantly reduce the shelf life of the test medium incorporating it.
In this connection, another form of the present invention provides a method of making a test medium for detecting at least one first type of biological entity and inhibiting a second type of biological entity from growing in the medium. The method includes the steps of combining desired substrates with a nutrient base medium; adding an inhibitor to the medium; and then sterilizing the medium by subjecting the medium to at least 100xc2x0 C. Because the inhibitor is added as an initial step, subsequent sterile addition of inhibitor is unnecessary.
In another form thereof, the present invention provides a test medium for detecting, identifying and qualifying or quantifying first, second and third biological entities. The test medium includes a nutrient base medium including ions of a salt. First and second chromogenic substrates and a nonchromogenic substrate are provided in the test medium. The first and second biological entities are responsive to the first and the second chromogenic substrates, respectively, and the third biological entity is responsive to the nonchromogenic substrate. Aggregations of the first biological entity present in the test medium are a first color, aggregations of the second biological entity present in the test medium are a second color, and aggregations of the third biological entity present in the test medium are substantially black.
In a preferred form, the inventive test medium accounts for the third biological entity being responsive to the first and/or the second chromogenic substrates in addition to the nonchromogenic substrate. In such event, aggregations of the third biological entity will nonetheless show as substantially black.
It should be appreciated that the use of a nonchromogenic substrate along with one or more chromogenic substrates synergistically increases the number of biological entities that can be detected and distinguished in a single medium and synergistically increases the possible color combinations for a given set of biological entities to be detected. Stated another way, including a nonchromogenic component as one of the substrates synergistically increases the degrees of freedom in selecting other substrates and corresponding colors for a test medium. This is so because an aggregation of the biological entity which is responsive to the nonchromogenic substrate will dependably show as substantially black. No combined color effects need be accounted for with the nonchromogenic substrates. For example, in a test medium including three chromogenic substrates and a nonchromogenic substrate, at least three combined color combination effects are avoided by using the one nonchromogenic component, as compared with using four chromogenic components.
The present invention, in another form thereof, provides a test medium capable of detecting, quantifying, and differentiating general coliforms and/or E. coli spp. under ambient light. The test medium comprises a nutrient based medium including a salt. A first substrate capable of forming a first water insoluble component of a first color in the presence of E. coli and the ions of the salt is provided in the medium. The first color is substantially black. A second substrate capable of forming a second water insoluble component of a second color in the presence of general coliforms is provided. The second color is visually distinguishable from the first color. Thus, colonies of E. coli present in the test medium are indicated by the first substantially black color and colonies of general coliforms are indicated by the second color.
In a preferred form of the above invention, the test medium further includes a third substrate capable of forming a third water insoluble component of a third color in the presence of Salmonella or Shigella spp. The third color is distinguishable from the first and second colors, whereby the test medium is capable of quantifying and/or differentiating E. coli, general coliforms and Salmonella or Shigella spp. Further, the substrates are selected such that general coliforms present in the test medium will also react with the third substrate to form a water insoluble component which includes the third color. Consequently, general coliform colonies are indicated in the test medium as a fourth color, the fourth color being a combination of the second color and the third color. The fourth color is visually distinguishable from the first and third colors. Still further, the substrates can be selected such that Aeromonas spp. form an insoluble component of the second color by reacting with the second substrate, but not the first and third substrates. Thus, in the inventive test medium, E. coli colonies will be generally black, general coliform colonies will be the fourth color, Aeromonas colonies will be the second color and Shigella or Salmonella colonies will be the third color.
In another form thereof, the present invention provides a method for detecting, quantifying and differentiating under ambient light general coliforms, E. coli, and at least one of the genera Aeromonas, Salmonella or Shigella in a test sample. The method comprises the steps of providing a nutrient base medium including first, second and third substrates. Each of the substrates is capable of forming a water insoluble component in the presence of at least one of general coliforms, E. coli Aeromonas, Salmonella or Shigella. The substrates are selected such that colonies of E. coli produced in the test medium are a first color, colonies of general coliforms produced in the test medium are a second color, and colonies of one of Aeromonas and Salmonella or Shigella produced in the test medium are a third color. Each of the colors are visually distinguishable. The test medium is inoculated with the test sample and then incubated. The test medium is then examined for the presence of first colonies having the first color, second colonies having the second color, and third colonies having the third color. The first colonies are E. coli, the second colonies are general coliforms, and the third colonies are one of Aeromonas, Salmonella or Shigella.
In a preferred form thereof, the inventive method further includes adding ions from a salt to the test medium to react with one or more of the substrates. In so doing, a precipitate is produced which shows as a substantially black color in the presence of the specific enzyme for that substrate. A preferred compound for forming the substantially black color in the presence of the ions of the salt consists of a xcex2-D-glucuronide. These compounds release an aglycone when hydrolized which forms a substantially black insoluble complex in the presence of ions.
In another preferred form of the inventive method, the method further comprises examining the test medium for the presence of fourth colonies having a fourth color, wherein the substrates are selected such that colonies of Aeromonas are the third color and colonies of Salmonella or Shigella are the fourth color, the fourth color being visually distinguishable from the first, the second and the third colors. More preferably, the substrates are selected such that the first color is substantially black, the second color is substantially blue-violet, the third color is substantially red-pink and the fourth color is substantially teal-green.
In another preferred form of the inventive method, the substrates are selected such that colonies of Aeromonas as well as colonies of Plesiomonas and Vibrios are indicated as the third color.
One advantage of the present invention is that it uses a nonchromogenic substrate along with one or more chromogenic substrates and thereby synergistically increases the degrees of design freedom in selecting colors for the inventive test medium. This is so because the chromogenic substrates do not interfere with the substantially black precipitate formed by the nonchromogenic substrate.
Another advantage of the present invention is that enables the quantification, identification and differentiation of four (4) different bacterial strains simultaneously in a single test medium using a single test sample, under ambient lighting. Subsequent tests with their concomitant extra time spent and extra costs are avoided. Of course, the inventive test medium of the present invention could also be used purely for qualitative purposes, as a mere presence/absence (P/A) test.
Yet another advantage of the present invention is that the substrates are selected such that the colors are easy to visually distinguish from one another without the need for UV light or other visual aids, other than, perhaps, magnification means. For example, in a preferred embodiment, E. coli colonies are clearly indicated by a precipitate having a substantially black color, general coliform colonies are indicated by a blue-violet color, Aeromonas colonies are indicated by a red-pink color, and Salmonella or Shigella colonies are indicated by a teal-green color. Because these colors are visually so distinct, confusion among the colors is greatly reduced as compared to prior art media.
Another advantage of the test medium of the present invention is its flexibility and ease of use. The incubation temperature is not critical as growth and differentiation of the organisms mentioned may occur within an optimum range. Therefore, resuscitation steps are avoided and inhibition of temperature sensitive strains does not occur. Also, inexpensive equipment may be used.
Yet another advantage of the present invention is that it intensifies the color distinction obtained in a test medium for identifying and differentiating E. coli from general coliforms. In a preferred test medium, E. coli colonies present a substantially black color, whereas general coliforms present a red-pink color, the distinction therebetween being much more apparent than in prior art test media. Confusion between the two colors is therefore greatly reduced.
Still another advantage of the present invention is that it enables the identification and differentiation of Aeromonas spp. from general coliforms. Prior art test media undesirably require using a cefsulodin inhibitor for preventing Aeromonas spp. from growing therein. However, the use of cefsulodin as an inhibitor requires an extra step in the process, viz., sterile addition of filter sterilized antibiotic, and is difficult to control. Further, the presence of cefsulodin significantly reduces the effective shelf life of the medium. Further, the use of an inhibitor, obviously, prevents the detection and quantitification of Aeromonas spp. Advantageously, with the present invention, Aeromonas spp. can be detected, quantified and differentiated from general coliforms in a single medium.
As a related advantage, if it is nonetheless desired to inhibit colonies of Aeromonas spp. from growing in the test medium, the present invention provides a superior means for doing so. Specifically, preferred forms of the present invention employ nalidixic acid as an inhibitor, which has been shown to have a far less deleterious effect to the shelf-life of the medium incorporating it. Further, nalidixic acid can be added as part of the initial medium formulation prior to sterilization, thereby avoiding a costly and difficult process step which is required with cefsulodin. Finally, nalidixic acid is much less expensive than cefsulodin.
Another advantage of the present invention is that it can provide a test medium for qualitative or quantitative testing. That is, the test media in accordance with the present invention can be used as mere presence/absence test devices, or can be used to quantify various biological entities showing as different colored colonies on the inventive test media.
The method and medium of the present invention allow the simultaneous detection, quantification, identification and differentiation of a variety of selected biological entities in a sample of mixed populations of biological entities. The inventive method and medium are particularly useful for the detection, quantification, identification and differentiation of E. coli and general coliforms, and further quantitative identification and differentiation of other selected biological entities, including Aeromonas, Salmonella, Shigella, Pseudomonas, and Vibrio bacterial species.
The method and test media incorporating the present invention utilize the fact that the enzymatic activity of biological entities and specifically of bacteria varies with the genus, and/or family of bacteria of interest. The method and test media incorporating the present invention further utilize the fact that various enzyme identifying substrate complexes can be used to identify specific enzymes with the production of distinctive colors. Significantly, the method and test media incorporating the present invention exploit the fact that chromogenic substrates present in a test medium do not interfere with the substantially black color produced by nonchromogenic substrates.
While nonchromogenic substrates are known in the art, per se, their distinct properties vis-xc3xa1-vis chromogenic substrates have been unrecognized. However, the behavior of a nonchromogenic substrate in a medium including a combination of chromogenic substrates is unique. To illustrate, aggregations of a biological entity which is responsive to two chromogenic substrates will typically show in a test medium as a combination of the the two colors produced upon cleavage of the two respective substrates. If three chromogenic substrates are involved, the combined color effect could be prohibitively difficult to predict and account for. Further, inherent variations in the amount of enzymes produced by particular strains of biological entities can result in different shades or hues of colors upon cleavage of the chromogenic substrates. Consequently, the colors can be difficult to distinguish for the lay person examining the test medium. Chromogenic substrates must therefore be chosen in view of the other chromogenic substrates planned for inclusion in a given test medium.
Such is not the case with the nonchromogenic components. While aggregations of biological entities which are responsive to chromogenic substrates in addition to nonchromogenic substrates may show in the test medium as having a colored or fluoroescent xe2x80x9chalo,xe2x80x9d such aggregations nonetheless appear substantially black and are therefore easy to identify. Unlike chromogenic substrates, multiple xe2x80x9cdegrees of freedomxe2x80x9d are achieved with the nonchromogenic components by not having to take into account combined color effects.
Using a nonchromogenic substrate enables a single test medium to differentiate four (4) different bacterial strains with four (4) visually distinguishable colors. The black color is superior in that it is difficult to mistake. Further, the substantially black pigmentation does not diffuse so that the location of the colonies is precisely known and the colonies can be accurately counted. The nonchromogenic substrates produce an insoluble chelated compound which is different than the dimer which is produced by the chromogenic substrates.
The inventive test medium and method allows not only a detection, quantification or qualitative identification and differentiation of general coliforms and E. coli, but also of Salmonella, Shigella and Aeromonas, as well as Plesiomonas and Vibrio. Plesiomonas and Vibrios species are determined but not differentiated from Aeromonas species as they are very closely related.
Biological entities, such as general coliforms, E. coli., etc., are herein referred to as being xe2x80x9cresponsivexe2x80x9d to certain chromogenic and nonchromogenic substrates. More specifically, a biological entity will predictably produce specific enzymes when the entity is present in a test medium such as the one described hereinbelow. These enzymes will selectively cleave chromogenic and nonchromogenic substrates. Upon cleavage, these substrates produce a color in the test medium. The mechanism for producing the color is different for chromogenic and nonchromogenic substrates, as described hereinbelow.
Microorganisms having xcex2-galactosidase activity include those commonly known by the designation xe2x80x9ccoliform.xe2x80x9d There are various definitions of xe2x80x9ccoliform,xe2x80x9d but the generally accepted ones include bacteria which are members of the Enterobacteriaceae family, and have the ability to ferment the sugar lactose with the evolution of gas and acid. Most coliforms are positive for both xcex1- and xcex2-galactosidase. That is, they produce both xcex1- and xcex2-galactosidases.
Microorganisms having xcex2-glucuronidase activity in addition to galactosidase activity primarily include most strains of Escherichia coli. That is, E. coli is positive for both xcex1- and xcex2-galactosidase as well as xcex2-glucuronidase.
The term xe2x80x9cgeneral coliformsxe2x80x9d as used in this application refers to coliforms other than the various strains of E. coli. These xe2x80x9cgeneral coliformsxe2x80x9d are gram-negative, non-spore forming microorganisms generally having xcex1- and xcex2-galactosidase activity (i.e., lactose fermenters), but not having xcex2-glucuronidase activity, and having the ability to ferment the sugar sorbitol.
For purposes of this specification, a xe2x80x9cchromogenic substratexe2x80x9d is a substrate which needs no additional chemicals present in the test medium upon hydrolysis for color production. That is, a chromogenic substrate is cleaved by the specific enzyme corresponding to that substrate to form a dimer with the color being concentrated in the area of cleavage of the substrate. Many chromogenic substrates are known in the art. For purposes of this specification xe2x80x9cchromogenicxe2x80x9d includes fluorogenic substrates. The products of fluorogenic substrates require ultraviolet (UV) light to be detected and are more soluble than preferred chromogenic substrates, so are therefore generally not preferred for use with the test media disclosed hereinbelow.
Certain substrates, referred to herein as xe2x80x9cnonchromogenic,xe2x80x9d produce a dark, substantially black precipitate in the presence of ions of a salt and enzyme activity. For example, 8-hydroxyquinoline-xcex2-D-glucuronide, when included in a medium along with a salt that produces ions, such as ferric ammonium citrate, will produce a substantially black precipitate in the presence of xcex2-glucuronidase produced by E. coli or other biological entities. More specifically, upon cleavage of the nonchromogenic substrate by the particular enzyme, a substantially black water-insoluble complex forms in the medium. The substantially black precipitate consists of the ferric ions and the aglycone released when the substrate is hydrolized by the glucuronidase from E. coli. This precipitate is a chelated compound which does not diffuse. Nor is the substantially black color susceptible to interference from chromogenic compounds present in the test medium.
For purposes of this specification a xe2x80x9cnonchromogenic substratexe2x80x9d means that a chemical in addition to those used with chromogenic components must be present in the test medium when the substrate is cleaved by its corresponding enzyme. The substantially black precipitate formed thereby is a combination of the substratexe2x80x94salt complex and is not a dimer as is formed by the xe2x80x9cchromogenic compounds.xe2x80x9d
For purposes of this specification, the expression xe2x80x9cunder ambient lightxe2x80x9d refers to the visible spectrum, i.e., colors which can be seen and distinguished with the naked eye. A colony present in a test medium which requires ultraviolet light to be seen, for example, would not fall under the definition xe2x80x9cunder ambient lightxe2x80x9d. However, it is to be understood that the term xe2x80x9cunder ambient lightxe2x80x9d includes using a magnification device, if necessary. Magnification can be especially helpful when counting numerous colonies. The term xe2x80x9cvisually distinguishablexe2x80x9d refers to two or more colors which can be differentiated under ambient light.
For purposes of this specification, the term xe2x80x9csubstantially blackxe2x80x9d includes dark brown to black, and also includes black with various colored halos, such as red-violet, green, fluorescent, etc.
For further purposes of this specification, color names recited herein are given as guidance, but it is to be understood that the color names are to be read broadly. That is, there can be overlap among the recited colors. This is because, as discussed, biological entities produce varying amounts of enzymes, which in turn affects the shade or hue of the resulting color.
The term xe2x80x9cxcex2-galactosidase substratexe2x80x9d as used herein refers to a xcex2-galactoside comprising galactose joined by xcex2-linkage to a substituent that forms a water insoluble colored compound when liberated by the action of xcex2-galactosidase on the substrate. Similarly, the term xe2x80x9cxcex1-galactosidase substratexe2x80x9d as used herein refers to xcex1-galactoside comprising galactose joined by xcex1-linkage to a substituent that forms a water insoluble colored compound when liberated by the action of xcex1-galactosidase on the substrate. The term xe2x80x9cxcex2-glucuronidase substratexe2x80x9d as used herein refers to a xcex2-glucuronide comprising glucuronic acid joined by xcex2-linkage to a substituent that forms a water insoluble colored precipitate when liberated by the action of xcex2-glucuronidase on the substrate.
The xcex1- and xcex2-galactosidase substrates and compounds and any other substrates described herein as well as the xcex2-glucuronidase substrates and compounds and any other substrates described herein may comprise carboxylate salts formed by reacting a suitable base with the appropriate galactoside or glucuronic carboxyl group. Suitable bases include alkali metal or alkaline earth metal hydroxides or carbonates, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and corresponding carbonates; and nitrogen bases such as ammonia, and alkylamines such as trimethylamine, triethylamine and cyclohexylamine.
Certain members of the family Enterobacteriaceae can be distinguished by the presence of xcex1-galactosidase activity in the absence of xcex2-galactosidase activity, or vice-versa. For example, most Salmonella and Shigella spp. are positive for xcex1-galactosidase, but negative for xcex2-galactosidase. Similarly, Aeromonas spp. can be distinguished from other members of the family Enterobacteriaceae by the presence of xcex2-galactosidase activity in the absence of xcex1-galactosidase activity. The method and medium incorporating the present invention are designed to take advantage of these distinguishing characteristics. For example, the specificity of enzyme activity for Salmonella and Aeromonas spp., as opposed to general coliforms, can be exploited, as illustrated below.
The method described herein is particularly suitable for the detection, quantification or qualitative identification and differentiation of the different classes of microorganisms described previously, viz., general coliforms, E. coli, Aeromonas and Salmonella and Shigella spp. Although the inventive method is particularly suitable for these particular microorganisms, it is not limited thereto. Instead, the techniques described herein have application to the identification and differentiation of a wide variety of biological entities.
That is, specific biological entities are xe2x80x9cresponsivexe2x80x9d to various substrates. More particularly, these biological entities predictably produce or contain known enzymes. Substrates, either chromogenic or nonchromogenic, can be selected which, in the presence of a particular enzyme(s), will form an insoluble component of a predictable and distinguishable color. Multiple substrates can be selected to simultaneously identify a plurality of distinct biological entities in a single test medium, aggregations of each distinct entity being identifiable by a separate, distinguishable color. Further, while the preferred embodiments disclosed herein distinguish all of the various aggregations present in a test medium under ambient light, as that term is defined herein, such is not necessary. For example, several substrates disclosed herein require the use of ultraviolet light for the aggregations present in the medium to be seen.
Table I lists various enzymes whose presence may be detected using certain of the substrates listed in Table II.
Specific substrate compounds applicable for use with the test medium of the present invention are available as follows:
5-bromo-4-chloro-3-indolyl-xcex2-D-galactopyranoside (X-gal) is a commercially available xcex2-galactosidase substrate that produces an insoluble precipitate having an approximately teal color when reacted upon by xcex2-galactosidase and is available from Biosynth International, Naperville, Ill.
6-chloro-3-indolyl-xcex2-D-glucuronide is a compound which produces an insoluble precipitate having a magenta color, the preparation of which is described in the aforementioned incorporated by reference U.S. Pat. No. 5,210,022 and is available from Research Organics, Cleveland, Ohio.
The compound 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide (X-gluc) is a commercially available xcex2-glucuronide that produces an insoluble precipitate having an approximately teal color when reacted upon by xcex2-glucuronidase. Similarly, indoxyl-xcex2-glucuronide is a similar compound, the preparation of which is described in the aforementioned article by Ley et al., in Can J. Microbiol., the disclosure of which is incorporated by reference.
Another suitable xcex2-galactoside is the compound 6-chloro-3-indolyl-xcex2-D-galactoside which produces an insoluble precipitate having a pink/magenta color, the preparation of which is described in the aforementioned U.S. Pat. No. 5,210,022.
Other suitable compounds applicable as substrates in the practice of the present invention are specified in U.S. Pat. No. 5,210,022, all of which are incorporated herein by reference.
The substrate 8-hydroxyquinoline-xcex2-D-glucuronide is a commercially available xcex2-glucuronide that, in the presence of metallic ions such as iron, produces an insoluble precipitate having a substantially black color when reacted upon by xcex2-glucuronidase and in the presence of other xcex1- or xcex2- galactoside substrates. 8-hydroxyquinoline-xcex2-D-glucuronide is available from Biosynth International, Naperville, Ill.
Further, a salt providing ions suitable for use with the present invention is ferric ammonium citrate, available from Sigma Chemical, St. Louis, Mo. The cyclohexenoesculetin substrates are described in James et al., Appl. and Envir. Micro. 62:3868-3870 (1996) and in the presence of ferric ions, produce an insoluble substantially black precipitate.
N-methyl-indolyl substrates such as N-methylhydroxy-xcex2-D-galactopyranoside are commercially available from Biosynth International, Naperville, Ill.
Nitrophenyl substrates, such as 2-nitrophenyl-xcex2-D-galactopyranoside, are commercially available from Biosynth International, Naperville, Ill. Similarly, nitroaniline compounds are available for synthesis through Sigma Chemical, St. Louis, Mo.
Other substrates producing a substantially black color include esculetin substrates such as cyclohexenoesculetin-xcex2-D-galactoside, which is described in James et al., Appl. and Envir. Microbiol. 62:3868-3870 (1996). Quinoline substrates, such as 8-hydroxyquinoline-xcex2-D-galactopyranoside and 8-hydroxyquinoline-xcex2-D-glucuronide are available through Biosynth International, Naperville, Ill.
Iodo-indolyl substrates, such as 5-iodo-3-indolyl-xcex2-D-galactopyranoside are available through Biosynth International, Naperville, Ill.
Several fluorescent substrates are suitable for use with the present invention. Coumarin substrates such as 4-methylumbelliferyl substrates and 5-trifluoromethylumbelliferyl substrates are commercially available from Biosynth International, Naperville, Ill. Also suitable are fluorescein substrates, rhodamine substrates, and resorufin substrates. No commercial source is known for these three substrates but components are available from Sigma Chemical, St. Louis, Mo.
While specific examples of substrates suitable for use with the present invention have been enumerated hereinabove, such is not to be construed as limiting the invention in any manner. Instead, one of ordinary skill in the art can use Table IV and V hereinbelow to identify a virtually limitless number of substrates.
The test medium is formed by combining the desired substrates with a nutrient base medium. The nutrient base medium can be any culture medium known in the art for providing the maintenance and reproduction of living cells. Generally, such media include nutrients, buffers, water, and sometimes a gelling agent. Possible gelling agents include agars, pectins, carrageenans, alginates, locust bean, and xanthins, among others.
The following is an example of the preparation of a test medium suitable for use in this invention. This example coincides with Example I, below.
The substrates 8-hydroxyquinoline-xcex2-D-glucuronide, 5-Bromo-4-chloro-3-indolyl-xcex1-D-galactopyranoside, and 6-Chloro-3-indolyl-xcex2-D-galactopyranoside are added in quantities of 250 mg/L medium; 70 mg/L medium; and 175 mg/L medium, respectively. The substrates are added directly to the hot (75xc2x0-85xc2x0 C.) medium (formula below) in a blender prior to sterilization.
Standard agar medium may be made by adding 15 gm of bacteriological quality agar gum to the following nutrient formula
and then sterilizing at 121xc2x0 C. for 15 minutes. The medium should be adjusted to result in a pH of 7.0. The sterilized agar medium is allowed to drop to a temperature of 45xc2x0 C. in a water bath and then the sterile solution containing the substrates prepared as described above is added. The medium is mixed thoroughly and poured into sterile petri plates at a volume of 20 ml/plate.
A pectin-based test medium may be prepared using the same steps described above except that 25 gm of low methoxyl pectin is used as the solidifying agent and the medium is poured at room temperature into petri plates containing a thin gel layer containing calcium ions which combine with the pectin to form a solid gel. A suitable pectin culture medium is described in U.S. Pat. Nos. 4,241,186 and 4,282,317, the disclosures of which are incorporated herein by reference. A pectin-based medium is preferred over a standard agar medium because it has the advantages of convenience and temperature independence for the user. The use of pectin media is well described and accepted as a result of AOAC collaborative studies and other published and in-house investigations.
A suitable pectin medium is commercially available from Micrology Laboratories, LLC under the trademark Easygel(copyright). Aqueous based medium without gelling agent is available from Micrology Labs, Goshen Ind., for use with membrane filters.
The test medium may be inoculated with a sample to be tested for the presence of microorganisms by any method known in the art for inoculating a medium with a sample containing microorganisms. For example, the sample to be tested may be added to the petri plates prior to adding the nutrient base medium (pour plate technique) or spread on the surface of the plates after they have cooled and solidified (swab or streak plate technique). Liquid samples may also be filtered through a micropore (0.45 micrometer size) membrane filter which is then placed on the surface of a solid medium or on a pad saturated with the medium.
The inoculated test medium is incubated for a sufficient time and at such a temperature for individual microorganisms present in the sample to grow into detectable colonies. Suitable incubation conditions for growing microorganisms in a medium are known in the art. Commonly, the test medium is incubated for about 24-48 hours at a temperature of about 30xc2x0-40xc2x0 C.
Unless inhibitors of the general microbial population are used, the general microbial population as well as general coliforms, E. coli, Aeromonas spp., and Salmonella spp. and Shigella spp. will grow in the incubated test medium. Because the precipitates formed are insoluble in the test medium, they remain in the immediate vicinity of microorganisms producing the various enzymes. As the microorganisms reproduce to form colonies, the colonies show as colony forming units having the color produced by the particular substrate.
For example, E. coli produces xcex2-galactosidase and xcex1-galactosidase, but, unlike general coliforms and Aeromonas spp., also produces xcex2-glucuronidase. Therefore, insoluble precipitates of each of the xcex2-galactosidase substrate, the xcex1-galactosidase substrate and the xcex2-glucuronide substrate are formed by the action of the respective enzymes such that colonies of E. coli show as a substantially black color, sometimes having a violet-blue halo therearound.
General coliforms produce xcex2-galactosidase and xcex1-galactosidase and consequently cleave both the xcex1-galactosidase and xcex2-galactosidase substrates. In the present example, the 5-Bromo-4-chloro-3-indolyl-xcex1-D-galactoside substrate produces a blue-green or teal color, whereas the 6-Chloro-3-indolyl-xcex2-D-galactoside produces a pink, or red-pink color. Thus, general coliform colonies will show as a blue-violet color, which is a combination of the colors produced by each of the xcex1- and xcex2-galactosides, respectively.
Significantly, however, it has been found that Aeromonas spp., which are closely related to coliforms, and give an almost identical biochemical test pattern, are xcex2-galactosidase positive and xcex1-galactosidase negative. That is, Aeromonas spp. will not hydrolize the xcex1-galactoside substrate. Therefore, Aeromonas colonies present in the test medium will show as colonies having the pink-red color produced by the xcex2-galactoside substrate.
Further, it has been found that most members of the genera Salmonella and Shigella are positive for xcex1-galactosidase, but negative for xcex2-galactosidase. That is, Salmonella and Shigella will not hydrolize the xcex2-galactosidase substrate. Therefore, colonies of Salmonella and Shigella present in the test medium will appear as a teal, or blue-green color produced by the xcex1-galactoside substrate. Occasionally, Shigella colonies will appear black with a blue-green halo since some strains of Shigella are positive for xcex2-glucuronidase, and some strains of Shigella will appear blue/purple since some unusual strains are positive for both xcex1-galactosidase and xcex2-galactosidase.
The substrates selected for the above example produce three distinct colors, and general coliforms are indicated by a fourth color which is a combination of two of the three colors. That is, E. coli colonies show as substantially black, general coliform colonies show as blue-violet, Aeromonas colonies show as red-pink, and Salmonella and Shigella colonies show as teal-green. While the individual shades of these colors may vary somewhat in the test medium due to factors such as varying enzyme production of the biological entities, it has been found that these four colors are distinct enough so that confusion amongst them is unlikely.
The colonies of each type of microorganism may be enumerated by counting the colonies or by other methods known in the art for enumerating microorganisms on a test plate. The number of colonies of each type indicates the number of microorganisms of each type originally present in the sample before incubation.
The method of the present invention does not require inhibitors. However, the medium may be made more selective for general coliforms and E. coli if desired by the addition of various compounds that are inhibitory to the general microbial population, but have little or no effect on coliforms. Following are some compounds which may be used: a) bile salts, about 0.3 g/liter, b) sodium lauryl sulfate, about 0.2 g/liter, c) sodium desoxycholate, about 0.2 g/liter, d) Tergitol 7, about 0.1 ml/liter. The use of one or more of these compounds reduces the background (non-coliform) microorganism presence and makes a less cluttered plate and eliminates the possibility of inhibition or interference by the non-coliform organisms in the sample. The use of certain antibiotics may accomplish the same result.
Cefsulodin is commonly used in currently available test media to inhibit Aeromonas spp. However, the use of cefsulodin as an inhibitor requires an extra step in the process, viz., sterile addition of filter sterilized antibiotic. This step is difficult to control. Further, the presence of cefsulodin significantly reduces the effective shelf life of the medium. It has been found that Nalidixic acid can be used instead of Cefsulodin to inhibit Aeromonas spp. with about the same efficacy. Nalidixic acid is preferable because it can survive the approximately 120xc2x0 C. temperature reached in autoclaving the test media. Therefore, unlike cefsulodin, nalidixic acid can be added to the test media as part of the initial media formulation prior to sterilization (see, preparation of test medium, above). It also follows that the resistance of the nalidixic acid to unfavorable environmental conditions will result in a longer shelf life for a medium containing it as compared to cefsulodin.
It is possible that the enzyme production of the general coliforms may be enhanced by the addition to the medium formulations of very small amounts of substances known as enzyme inducers. One specific inducer for xcex2-galactosidase is available and is known chemically as isopropyl-xcex2-thiogalactopyranoside. Adding approximately 100 mg/liter of medium has a positive and noticeable effect on the speed of enzyme production for some species of coliforms. Other enzyme inducers are available and may be added to media formulations if enhanced enzyme production is deemed helpful.